von Willebrand factor links primary hemostasis to innate immunity

The plasma multimeric glycoprotein von Willebrand factor (VWF) plays a critical role in primary hemostasis by tethering platelets to exposed collagen at sites of vascular injury. Recent studies have identified additional biological roles for VWF, and in particular suggest that VWF may play an important role in regulating inflammatory responses. However, the molecular mechanisms through which VWF exerts its immuno-modulatory effects remain poorly understood. In this study, we report that VWF binding to macrophages triggers downstream MAP kinase signaling, NF-κB activation and production of pro-inflammatory cytokines and chemokines. In addition, VWF binding also drives macrophage M1 polarization and shifts macrophage metabolism towards glycolysis in a p38-dependent manner. Cumulatively, our findings define an important biological role for VWF in modulating macrophage function, and thereby establish a novel link between primary hemostasis and innate immunity.


Field-specific reporting
Please select the one below that is the best fit for your research. If you are not sure, read the appropriate sections before making your selection.

Life sciences
Behavioural & social sciences Ecological, evolutionary & environmental sciences For a reference copy of the document with all sections, see nature.com/documents/nr-reporting-summary-flat.pdf

Life sciences study design
All studies must disclose on these points even when the disclosure is negative. The RNA-Seq data files have been submitted to NCBI's Gene Expression Omnibus and are stored under accession number GSE205365 which has been made publicly available.
All datasets used and/or analyzed during the study have been included in the attached Source data file.
n/a n/a n/a n/a No power calculations were performed for in vitro experiments. We used standard in vitro sample size of n=3 biological for BMDC derived from mice. Biological replicates of at least 3 were used for BMDC experiments. For metabolic assay studies each biological replicate had at least 3 technical replicates. Each figure contains full details regarding sample numbers for the related figure.
No data were excluded from analyses.
Experiments were performed a minimum of 3 times independently. All attempts at replication were successful. Each figure legend contains details of experimental replicates for the related figure.
Both male and female littermate mice of C57Bl/6J background were used at 6-10 weeks of age for WT BMDM generation. Our study did not involve any experiments where randomization would increase the reliability of the results.
Blinding was not required in this study. All data were recorded and analysed under the same experimental conditions to exclude any bias. Note that full information on the approval of the study protocol must also be provided in the manuscript.

Flow Cytometry
Plots Confirm that: The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).
The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).
All plots are contour plots with outliers or pseudocolor plots.
A numerical value for number of cells or percentage (with statistics) is provided.

Methodology
Sample preparation All cell lines tested negative for mycoplasma None Adult 8 to 12 weeks old C57BL/6J strain mice were used in all experiments. Animals were housed in a specific pathogen-free facility in individually ventilated and filtered cages under positive pressure.
No wild animals were used in the study.
Both male and female littermate mice of C57Bl/6J background were used at 6-10 weeks of age for wild type BMDM generation. Preliminary experiments did not show any differences according to sex.
No field collected samples were used in the study.
All animal experiments were approved by the Animal Research Ethics Committee, Trinity College Dublin, and under license from the Ireland Health Products Regulatory Authority (AE19127/P060). All procedures conformed to the Directive 2010/63 EU of the European Parliament.
VWF Binding Differentiated THP1 cells were detached from petri dishes by continuous ice cold PBS pipetting and subsequently divided between treatments (5x105 cells/flow tube). Alternatively, non PMA treated suspension monocytic THP1 cells were used. VWF variants of interest were incubated with THP1 macrophages in binding buffer (Hanks balanced salt solution supplemented with HEPES (10mM), MnCl2 (1mM) and CaCl2 (1mM ) ± ristocetin (Helena, UK 1.5mg/ml) where indicated) for 30 minutes at room temperature. Cells were washed once in 2ml of ice cold binding buffer. FC-receptors were blocked for 10 minutes on ice. Where applicable, THP1 cells were washed with 2ml of binding buffer between and after 30 minute incubations (on ice) with primary and secondary antibodies. Following staining, cells were fixed in binding buffer containing 1% PFA for 10 minutes on ice.
Human primary macrophages were differentiated from buffy coat isolated CD14High monocytes on 48 well low adherent plates (Starstedt, Germany) and detached by gentle scraping with the barrel of a 1ml syringe. Cells were pooled and equally divided between treatment flow tubes (2x105-1x106). VWF was incubated for 30min at room temperature and cells were washed with room temperature RPMI Phenol red free, supplemented with CaCl2 (1mM ). FC-receptors were blocked for 10 minutes, and without washing cells were incubated with anti-vwf for 30mim. Cells were washed RPMI Phenol red free, supplemented with CaCl2 (1mM ) before and after a 30min incubation with Anti-Rabbit Alexa Fluor 488. Following staining, cells were fixed in binding 1% PFA for 10 minutes on ice.
M1 and M2 Populations BMDM were washed with RPMI++ media, lifted by lite scraping with the barrel of a 1ml syringe. Fc receptors were block for 10min on ice, and without washing cells were stained(and IgG control) for 30 minutes on ice. Cells were washed in RPMI++ then fixed in 1% PFA for 10 minutes on ice and washed again.

Murine Lavage Fluid
Lavage fluid was harvested and cells were washed in 5% FBS in RPMI phenol red free and subsequently Fc blocked was added for 10 min before staining. Without washing cells were stained (and IgG controls) for 30min in 5% FBS in RPMI phenol red free on ice. Cells were washed in 5% FBS in RPMI phenon red free, then fixed in 1% PFA for 10 minutes. Cells were washed once before running.